Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells (Treg). We have demonstrated that the regulatory T cells can be easily identified in normal lymphoid tissues by expression of CD4, the interleukin-2 receptor alpha chain (CD25), and the transcription factor, FoxP3. Transfer of CD4+CD25-Foxp3- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+Foxp3+ T cells. The major goals of this project are to define the function and mechanism of action of Treg cells in vivo: (1) We have previously demonstrated that the target antigen in autoimmune gastritis (AIG) is the H+/K+ATPase, the proton pump of the gastric parietal cell. Our recent studies have demonstrated that stimulation of naive polyclonal T cells in the presence of TGF-beta results in the induction of Foxp3 expression and T regulatory activity in vitro. We have demonstrated that naive H+/K+ ATPase-specific CD4+CD8-Foxp3- thymocytes from TCR transgenic mice can be easily expanded and converted to FoxP3+ T cells when activated in the presence of TGF-beta. Most importantly, these autoantigen-specific iTregs were able to potently inhibit the development of AIG. iTregs prevented the priming and expansion of the effector T cells very early after cell transfer and appeared to decrease the stimulatory capacity of dendritic cells (DC) presenting endogenous autoantigen rather than by competing with the effectors for antigen or by acting directly with the effector cells to prevent their interaction with the DC. Thus, iTregs stop the autoimmune reaction before it even starts. The iTregs specifically reduced the expression of the co-stimulatory molecules CD80 and CD86 on the surface of autoantigen presenting DC in vitro. Taken together, these data demonstrate that it is possible to convert potential autoantigen-specific effector T cells to potent autoantigen-specific iTregs. A similar approach might be applicable to the treatment of autoimmune disease in man. (2) CD4+Foxp3+ can be generated from any nave antigen-specific CD4+Foxp3- T cell in vitro in unlimited numbers by TCR stimulation in the presence of TGF-beta and IL-2. We have developed a rapid in vivo model for measuring the suppressive activity of TGF-beta-Treg. TCR transgenic CD4+Foxp3- are stimulated in vitro for 4-5 days in the presence of TGF-beta. They are then co-transferred into normal recipients with congenically marked, CFSE-labeled naive TCR transgenic responder cells. The recipients are then immunized with peptide in complete Freunds adjuvant and the responder population in draining lymph nodes analyzed one week later. In the presence of antigen-specific TGF-beta-Treg, expansion of the responder cells is inhibited by >90%. In addition, differentiation of the responders to either Th1 or Th17 cells is also completely blocked. The TGF-beta-Treg proliferate in response to antigen and maintain Foxp3 expression. Activation of antigen presenting cells (APC) by TLR-ligands in the mycobacteria does not abrogate suppression as identical results were observed with complete or incomplete Freunds adjuvant. Suppression was also not reversed by treatment with anti-IL-10R. Transfer of 50-fold more polyclonal Treg had no effect on responder cell proliferation confirming the enhanced suppressive capacity of antigen-specific Treg. This model should allow us to readily determine in an immunocompetent environment the cellular target (s) of the antigen-specific TGF-beta-Treg, and the cellular/molecular basis for their suppressive effects in vivo. (3) The concept of Th1/Th2 dichotomy has been challenged by the discovery of Th17 cells as major players in both systemic and organ-specific autoimmunity. We have compared the ability of fully differentiated Th1, Th2, and Th17 effector T cells specific for the gastric parietal cell (PC) antigen, H/K ATPase, to induce AIG and have analyzed their susceptibility to Treg-mediated suppression. Nave CD4+25- thymocytes from mice expressing a transgenic TCR specific for the H/K ATPase alpha-chain529-541 peptide were differentiated in vitro into Th1, Th2 and Th17 T-effectors. Each cell line was injected into immuno-incompetent nude mice, either alone, or with freshly isolated polyclonal CD4+25+Foxp3+ Tregs. Recipient mice were then monitored for the presence of anti-PC antibodies, mucosal infiltration, and PC destruction. Prior to transfer, each cell line produced its respective signature cytokines (IFNgamma, IL-4, IL-17). All cell lines preferentially proliferated in the gastric lymph nodes (gLN) and induced AIG. The inflammatory infiltrates in Th1-injected mice almost exclusively consisted of CD3 lymphocytes, while Th2-induced infiltrates also included clusters of eosinophils and macrophage giant cells. Th17-induced infiltrates were made up of large numbers of eosinophils. Mice with AIG have enlarged gLN and the largest gLN were observed in Th17 recipients. The gLN of Th17 recipients contained more (2-3X) transgenic effector cells than recipients of Th1 and Th2 cells, as well as an increased number of CD19+ B cells. Co-transfer of Treg resulted in a reduction of anti-PC-Ab titers and disease severity. However, 6 weeks after transfer, suppression was more effective in the Th1 (84% of the mice protected) than inTh2 group (70%). In the Th17 group, slight suppression was effective after 4 weeks, but no suppression was seen after 6 weeks. Fully differentiated Th1, Th2, and Th17 effector cells can all induce AIG with much more severe disease seen in Th17 recipients. Tregs can prevent autoimmunity mediated by Th1 and Th2 differentiated cell lines, but Th17induced disease is highly resistant to suppression. (4) The approach we have taken to understanding the regulation of the immune response to chronic viral infections has been to determine the potential role that Tregs may play in lymphocytic choriomeningitis virus (LCMV) infection in mice. Our initial approach has been to compare the phenotypic changes in Tregs during the natural course of acute (strain Armstrong ) and chronic (strain Clone 13) LCMV infection. During clone 13 infection, we have identified a subpopulation of Tregs expressing TCR Vbeta5 that increase in frequency among Foxp3 positive cells from 7% in uninfected and Armstrong infected mice to 25% in clone 13 infected mice and peak at around 20 days post infection. The increase in frequency of Vbeta5+ Tregs is due to the preferential expansion of this population early in infection. No change in the frequency of Vbeta5+, CD4+, Foxp3- T cells was noted during either Armstrong or clone 13 infections. The Vbeta5+, Foxp3+ Tregs also express markers that have been associated with Treg activation in vivo including CD103 and granzyme B. The antigen specificity of the Vbeta5+ Treg population is as yet undetermined. They may recognize a viral superantigen or an endogenous superantigen that is activated by LCMV infection. Alternatively, Vbeta5+ population may recognize a unique LCMV peptide(s) through conventional interaction of TCR and peptide/MHC. To address this possibility, we will screen the Vbeta5+ population with the entire LCMV proteome using overlapping 15-mer peptides that span all four LCMV proteins. Finally, we are working to determine the functional significance of the expanded Vbeta5+ Tregs using mice that can be depleted of Tregs, and various adoptive transfer models. All together, we hope these approaches will contribute to our understanding of how Tregs may contribute to the immunoregulation of chronic LCMV infection.